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Immunofluorescence by Zenon
Author | Akihiro Harada | ||
---|---|---|---|
Affiliation | Department of Cell Biology, Graduate School of Medicine, Osaka University | ||
Contact | inquiry about this protocol | ||
Home Page | http://www.med.osaka-u.ac.jp/pub/acb/eng.html | ||
Keyword | |||
Published | 2015-08-26 | Last Update | 2015-08-26 |
View | 25 | download PDF | 7 |
Summary
Methods
Detail *Click photo to see a large size.
- Wash the sections with PBS once
- Block the nonspecific staining by treating the sections in 5%Normal donkey Serum/PBS
- Treat the sections with diluted primary antibody in 5%Normal donkey Serum/PBS, 37C 1hr
- Wash the sections with PBS several times
- Block the nonspecific staining by treating the sections in 5%Normal donkey Serum/PBS
- Treat the sections with diluted secondary antibodies in 5%Normal donkey Serum/PBS, 37C 30min
- Wash the sections with PBS several times
- Block the nonspecific staining by treating the sections in 5%Normal Goat Serum/PBT(0.1%TritonX-100/PBS)
- Treat the sections with diluted tertiary antibodies (labeled by Zenon) in PBT, RT 1hr
- Wash the sections with PBS several times
- Postfix the sections with 4%PFA/PBS,RT 15min
- Wash the sections with PBS once
- Embedding
- Protocol for making tertiary antibody
1) Mix antibody solution 10μℓ+Zenon Comp A 10μℓ, RT, more than 5min(less than a few days)
2) Add Zenon Comp B 10μℓ to mixture 1(total 30μℓ), RT 5min
3) Add PBT 70μℓ to mixture 2 (total 100μℓ)